Identification of Thermal Conduits That Link the Protein–Water Interface to the Active Site Loop and Catalytic Base in Enolase

Abstract

We report here on the salient role of protein mobility in accessing conformational landscapes that enable efficient enzyme catalysis. We are focused on yeast enolase, a highly conserved lyase with a TIM barrel domain and catalytic loop, as part of a larger study of the relationship of site selective protein motions to chemical reactivity within superfamilies. Enthalpically hindered variants were developed by replacement of a conserved hydrophobic side chain (Leu 343) with smaller side chains. Leu343 is proximal to the active site base in enolase, and comparative pH rate profiles for the valine and alanine variants indicate a role for side chain hydrophobicity in tuning the pK a of the catalytic base. However, the magnitude of a substrate deuterium isotope effect is almost identical for wild-type (WT) and Leu343Ala, supporting an unchanged rate-determining proton abstraction step. The introduced hydrophobic side chains at position 343 lead to a discontinuous break in both activity and activation energy as a function of side chain volume. Hydrogen−deuterium exchange mass spectrometry (HDX-MS) experiments were performed as a function of time and temperature for WT and Leu343Ala, and provide a spatially resolved map of changes in protein flexibility following mutation. Impacts on protein flexibility are localized to specific networks that arise at the protein−solvent interface and terminate in a loop that has been shown by X-ray crystallography to close over the active site. These interrelated effects are discussed in the context of long-range, solvent-accessible and thermally activated networks that play key roles in tuning the precise distances and interactions among reactants.

Hydrogen/Deuterium Exchange Measurements May Provide an Incomplete View of Protein Dynamics: a Case Study on Cytochrome c

“…The sparsely populated 5- and 4-coordinate Fe­(III) states are undetectable in traditional spectroscopic experiments, where signals are dominated by the 6-coordinate ground state. HDX methods are unique in this regard because they report on rare excursions to NH OPEN conformers that have very low Boltzmann probabilities. ,,,− In summary, Fe­(III) cyt c shows enhanced HDX compared to the Fe­(II) protein because 4-coordinate conformers are more highly populated in the Fe­(III) state, as dictated by its weaker coordination bonds. ,− These findings are in line with “scenario ( ii )” that we outlined above.…”

“…The occupancy of each conformer is determined by its free energy . Fluctuations between the native ground state and alternative conformations are believed to mediate enzyme catalysis, − allosteric regulation, − ligand binding, , aggregation, degradation, and so on. However, details of these dynamics–function relationships remain poorly understood.…”

“…HDX-MS is being used for numerous applications, from fundamental biophysics to the characterization of protein drugs. Most HDX-MS studies are conducted in a comparative fashion by examining a protein under different conditions, for example, unmodified versus chemically altered. ,,,− …”

“…HDX for most native proteins proceeds in the EX2 limit ( k cl ≫ k ch ), where each NH visits the open state numerous times before it is deuterated, resulting in an HDX rate constant k HDX = K op k ch , with K op = k op / k cl . , Under EX2 conditions, the opening/closing frequency does not affect k HDX as long as K op remains constant. , Opening/closing in eq involves short-lived unfolding/refolding transitions that can represent local, subglobal, or global events. ,, Protein regions that undergo fast deuteration (large K op ) are considered to be “dynamic”, while slow deuteration (small K op ) signifies “rigid” elements. ,,,− The very low equilibrium populations of NH OPEN conformers are undetectable by conventional methods, where signals are dominated by the native state …”

Temporal and Spatial Resolution of a Protein Quake that Activates Hydrogen Tunneling in Soybean Lipoxygenase

“…A revised working model of protein dynamics, Fig. 6, begins with the growing body of experimental evidence for protein embedded networks (18)(19)(20)(21)(22) that provide pathways for heat activation in thermally initiated enzyme reactions. The principle of conformational selection in protein function, as conceptualized by Frauenfelder for ligand binding dynamics in myoglobin (92), and developed by Kern and co-workers for catalyzed reactions (90,91) can be readily integrated with this feature, via substrate induced shifts in the conformational ensemble of enzyme-sub-state complexes toward configurations that place the reactive components of an enzyme active site in close proximity to the region of protein capable of efficient heat transmission.…”